Field of the Invention
This invention relates, e.g., to in vitro methods for joining (recombining) double stranded DNA molecules via a region of homology. In one embodiment, a plurality of DNA molecules are joined into a longer DNA molecule in a predefined order and orientation.
Background Information
Homologous recombination of linear double stranded DNA has long been known to be crucial for the repair of double stranded DNA breaks. In most organisms, the initiation of homologous recombination requires the action of an exodeoxyribonuclease. The single stranded DNA fragment generated can then pair with homologous sequence on other DNA molecules to complete the recombination. Although homologous recombination has been intensely studied, the mechanism involved is still not fully understood. The most efficient homologous recombination system has been discovered in Deinococcus radiodurans, which can survive 15,000 Gy of ionizing radiation, while doses below 10 Gy are lethal to almost all other organisms (Daly et al. (1996) J. of Bacteriology 178, 4461-4471). However, due to the complexity of the D. radiodurans genome, it is extremely difficult to pinpoint the proteins involved in the homologous recombination process.
Homologous recombination has also been demonstrated in the enterobacteria phage T7 system, the efficiency of which can be more than 50% (Lai et al. (1998) J. of Bacteriology 180, 6193-6202). T7 phage contain only 56 genes which encode 59 proteins, and therefore would be a more suitable system to isolate proteins involved in homologous recombination. In the T7 genome, genes that are involved in similar functions are normally clustered together. It has been reported that the early genes from gene 1.3 ligase to gene 6 exonuclease may be important in recombination (Lee et al. (1983) J. of Virology 48, 647-653; Lai et al. (1998), supra; Lai et al. (2000) Molecular Microbiology 36, 437-466; Yu et al. (2001) J. of Bacteriology 183, 1862-1869). However, it is not known whether host proteins are also important in this process.